We are proposing extensive studies on the structure and metabolism of collagen and its biosynthetic precursor procollagen by cultured human skin fibroblasts in control and disease states. The overall goal of these studies is to precisely define the molecular defects which may occur in the biochemistry of collagen in diseases affecting skin and other connective tissues. The studies include a variety of both acquired and inherited diseases in which previous investigations by us and others have revealed connective tissue abnormalities but the exact nature of the collagen aberrations is currently unknown. The main emphasis of these studies will be on two distinct disease entities, progressive systemic sclerosis and the Ehlers-Danlos syndrome. Cells from such patients will be subjected to in-depth analyses using a variety of techniques, including the recombinant DNA technology. In case of the Ehlers-Danlos syndrome, we attempt to define structural mutations in collagen by employing three main strategies: a) examination of mRNA and cloned cDNA molecules; b) detection of mutated sequences by digestion of mRNA-cDNA hybrids with S1 nuclease; c) examination of genomic DNA by restriction enzyme mapping. In case of scleroderma, the analyses include the assay of type I and III collagen specific mRNA levels and activities in cultured fibroblasts. We also plan to determine the copy number of the corresponding genes as an indication of possible gene amplication. We expect that the experiments proposed here will provide definitive information about mutations in the structural genes in type I procollagen in the Ehlers-Danlos syndrome. Such data will help up to define the normal structure-function relationship of type I collagen in skin and other tissues, as it pertains to specific domains and amino acid sequences in the protein. The studies of scleroderma, and related skin disorders, are expected to yield precise information on the underlying molecular mechanisms of these conditions. Thus, the results will provide us with knowledge which is highly useful for the development of rational treatment modalities for these conditions. In addition, the proposed studies, such as those exploring gene amplification in scleroderma, may provide results leading to a new understanding of the arrangement and functional aspects of human genes.